WO2022127886A1 - 利用生物多酶偶联法制备l-草铵膦的方法 - Google Patents

利用生物多酶偶联法制备l-草铵膦的方法 Download PDF

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WO2022127886A1
WO2022127886A1 PCT/CN2021/139009 CN2021139009W WO2022127886A1 WO 2022127886 A1 WO2022127886 A1 WO 2022127886A1 CN 2021139009 W CN2021139009 W CN 2021139009W WO 2022127886 A1 WO2022127886 A1 WO 2022127886A1
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amino
reaction
glufosinate
transaminase
product
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PCT/CN2021/139009
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French (fr)
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魏东芝
王华磊
吴承骏
刘清海
罗中华
张长雷
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永农生物科学有限公司
华东理工大学
宁夏永农生物科学有限公司
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Publication of WO2022127886A1 publication Critical patent/WO2022127886A1/zh

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    • C—CHEMISTRY; METALLURGY
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    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids

Definitions

  • the present application relates to the field of biotechnology, and in particular to a method for preparing L-glufosinate-ammonium using a biological multi-enzyme coupling method.
  • Glufosinate-ammonium also known as bialaphos, glufosinate, the trade names include Baoshida, Besutun, etc., the English name is phosphinothricin (abbreviated as PPT), the chemical name is 2-amino-4-[hydroxy (methyl) ) Phosphono]butyric acid
  • PPT phosphinothricin
  • glufosinate-ammonium The mechanism of action of glufosinate-ammonium is that after glufosinate-ammonium acts on plants, it can inhibit glutamine synthase, thereby interrupting the reversible reaction of glutamate in plants, causing metabolic disorders, accumulation of excess ammonia, poisoning plants, and at the same time causing plants to fail. Synthesis of chlorophyll, the disintegration of chloroplasts, the photosynthesis of plants will be inhibited, resulting in plant death.
  • Glufosinate-ammonium is mainly used in orchards, potato fields, non-arable land, etc. to control annual and perennial grass and dicot weeds, such as crabgrass, foxtail, wild wheat; perennial grass weeds and sedges, such as fescue, Duck sprouts etc.
  • glufosinate-ammonium There are two optical isomers of glufosinate-ammonium, namely L-glufosinate-ammonium and D-glufosinate-ammonium, but only the L-type has herbicidal activity, and it is easily decomposed in the soil and is less toxic to humans and animals. Weeding has a wide spectrum and little damage to the environment.
  • glufosinate-ammonium sold on the market is generally a racemic mixture. If glufosinate-ammonium products can be used in the form of pure optical isomers of L-configuration, the usage amount of glufosinate-ammonium can be significantly reduced, which is of great significance for improving atom economy, reducing use cost and reducing environmental pressure.
  • the chemical method includes chemical separation method and chemical synthesis method.
  • the chemical resolution method is to separate the racemic D,L-glufosinate or its derivatives synthesized by the external chemical method by chiral resolution reagents, so as to obtain optically pure L-glufosinate.
  • Hoechst Company reported the use of quinine as a resolving agent to split glufosinate-ammonium racemate, and realized the separation of D- and L-glufosinate-ammonium (US patent US5767309).
  • this process has disadvantages such as the need to use a chiral resolution reagent, the need for racemization of D-glufosinate-ammonium, and the need for multiple resolutions, making it difficult for large-scale industrial production.
  • the chemical synthesis method uses natural chiral amino acid or asymmetric method to synthesize L-glufosinate.
  • Hoechst Company reported the use of L-glutamic acid or L-aspartic acid as a chiral source to synthesize L-glufosinate (Hoffmann MG, Zeiss H J. A novel and convenient route to L-homoserine lactones and L-phosphinothricin from L-aspartic acid [J]. Tetrahedron Letters, 1992, 33(19): 2669-2672. and European Patent EP0530506).
  • the amino protection and deprotection process of this process is complicated and involves many steps, which are mostly found in laboratory studies.
  • biocatalysis has the advantages of mild reaction conditions, high stereoselectivity, and high yield, and is an important trend for the industrial preparation of L-glufosinate-ammonium.
  • L-glufosinate Most of the methods for preparing L-glufosinate by biocatalysis currently use 2-oxo-4-(hydroxymethylphosphinyl)butyric acid (PPO) as the substrate, and then asymmetrically synthesize L-glufosinate through ammoniation reaction.
  • PPO 2-oxo-4-(hydroxymethylphosphinyl)butyric acid
  • phosphine Among them, CN1349561A describes the synthesis of L-glufosinate from PPO through transamination by using aspartate as an amino donor and using the screened aspartate aminotransferase (Asp-TA).
  • Patent CN105603015B describes a method for producing L-glufosinate using aminotransferase with L-alanine as amino donor.
  • This process can completely convert 100 mM PPO into L-Glufosinate-ammonium, but in order to inhibit the reverse reaction, the dosage of alanine is too high, and 300 mM L-alanine still exists in the system at the end of the reaction, which causes difficulties for subsequent separation and purification , while the reaction temperature is as high as 50 ° C, the energy consumption is high, which is not conducive to production.
  • PPO under the mediation of amino acid dehydrogenase, using inorganic ammonia as a donor, PPO is reductively aminated to prepare L-fine glufosinate-ammonium, such as CN106978453A. Inorganic ammonia is used in the reaction process, and the conversion rate can reach 99%. Above, however, this process requires an additional coenzyme regeneration system, which increases the difficulty of separation and purification.
  • the present application provides a method for preparing L-glufosinate-ammonium by utilizing a biological multi-enzyme coupling method for redox asymmetric preparation, which uses D,L-glufosinate as a raw material, and obtains L-glufosinate through a multi-enzyme catalytic system.
  • the method has high raw material conversion rate, simple separation and purification process, high product yield, low production cost and easy industrialization.
  • the application provides a method for preparing L-glufosinate, comprising:
  • step b) in the presence of (S)-transaminase and amino donor, transamination reaction of 2-carbonyl-4-[hydroxy(methyl)phosphono]butyric acid obtained in step a) to obtain L-glufosinate and the deamination product of the amino donor.
  • step a racemic D,L-glufosinate-ammonium is used as a substrate, and (R)-transaminase converts D-glufosinate to D-glufosinate in the presence of a small amount of amino acceptor such as pyruvate (no need to separate after the reaction).
  • the amino group is transferred to the amino acceptor and deaminated to generate 2-carbonyl-4-(hydroxymethylphosphono)butyric acid (PPO), while L-glufosinate does not participate in the reaction and is completely retained.
  • PPO 2-carbonyl-4-(hydroxymethylphosphono)butyric acid
  • Transaminase (Amine Transaminase, ATA, EC 2.6.1.X) belongs to the class of transferases, which catalyze the transfer of an amino group on an amino donor (amino acid or simple amine) to a prochiral acceptor ketone to obtain a chiral amine
  • a class of enzymes with by-products ketones or ⁇ -keto acids, the reactions catalyzed are reversible.
  • Transaminases can be further divided into ⁇ -transaminases and ⁇ -transaminases (EC2.6.1.1). The ⁇ -transaminase reaction process can be divided into two steps.
  • the first reaction is to transfer the amino group on the amino donor to the carbonyl of PLP under the action of ⁇ -transaminase, thereby forming pyridoxamine 5-phosphate (PMP) and pyridoxamine 5-phosphate (PMP).
  • PMP pyridoxamine 5-phosphate
  • PMP pyridoxamine 5-phosphate
  • PMP pyridoxamine 5-phosphate
  • PMP pyridoxamine 5-phosphate
  • the ketone corresponding to the amino donor; the second step of the reaction also transfers the amino group on the PMP to the amino acceptor under the action of ⁇ -transaminase, and the PMP is converted into PLP to realize the cycle.
  • Stereoselectivity of transaminases can be determined in racemic resolution of racemic mixtures of substrates containing chiral centers.
  • ⁇ -aminotransferases can be divided into "(R)-aminotransferases” ((R)-amine transaminase) and (S)-aminotransferases ((S)-amine transaminase).
  • (R)-transaminase may be an enzyme that preferentially induces a transamination reaction of D-glufosinate from racemic D,L-glufosinate in the presence of a ketone substrate such as pyruvate .
  • the (R)-transaminase can be any enzyme known in the art having (R)-transaminase activity.
  • the (R)-transaminase is any one selected from the group consisting of: APH1 (eg, NCBI SEQ ID NO: WP_015938787.1), HEA-2 (eg, NCBI SEQ ID NO: ABX05998.1), TSP -1 (eg NCBI serial number is WP_013128145.1), DEP-2 (eg NCBI serial number is WP_013615256.1) and MPH (eg NCBI serial number is WP_013863226.1).
  • the (R)-transaminase is APH1.
  • the (R)-transaminase is from Pseudarthrobacter chlorophenolicus, eg, APH1 from Pseudarthrobacter chlorophenolicus.
  • the amino acid sequence of the (R)-transaminase has at least 70%, 80%, 90%, 91%, 91%, 93%, 94%, 80%, 90%, 91%, 91%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or 100% identity.
  • the nucleotide sequence of the (R)-transaminase is at least 60%, 70%, 80%, 90%, 91%, 91% of the nucleotide sequence shown in SEQ ID No.
  • amino acid sequence of the (R)-transaminase is SEQ ID No. 1.
  • nucleotide sequence of the (R)-transaminase is SEQ ID No. 2.
  • the amino acceptor in step a) is pyruvate, methyl pyruvate or ethyl pyruvate. In some preferred embodiments, the amino acceptor in step a) is pyruvate.
  • the amino addition product of the amino acceptor in step a) is D-alanine.
  • a catalytic enzyme capable of converting the amino addition product of the amino acceptor to the amino acceptor is additionally present in step a).
  • the amino-acceptor is converted into the amino-acceptor by converting the amino-acceptor into the amino-acceptor under the action of the catalytic enzyme, so that the in-situ regeneration of the amino-acceptor is achieved while the reaction system has better catalytic efficiency, The use of a large number of amino acceptors is avoided.
  • the catalytic enzyme capable of converting the amino addition product of the amino acceptor to the amino acceptor in step a) is D-amino acid oxidase, which converts D-alanine to acetone acid.
  • the D-amino acid oxidase (EC 1.4.3.3) described herein may be any enzyme known in the art having D-amino acid oxidase activity.
  • the D-amino acid oxidase is from Rhodotorula sp.
  • the D-amino acid oxidase is from Rhodotorula sp. CCFEE 5036.
  • the amino acid sequence of the D-amino acid oxidase has at least 70%, 80%, 90%, 91%, 91%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or 100% identity.
  • the nucleotide sequence of the D-amino acid oxidase is at least 60%, 70%, 80%, 90%, 91%, 91% of the nucleotide sequence shown in SEQ ID No. 4 , 93%, 94%, 95%, 96%, 97%, 98% or 99% or 100% identity.
  • the amino acid sequence of the D-amino acid oxidase is SEQ ID No.3.
  • the nucleotide sequence of the D-amino acid oxidase is SEQ ID No.4.
  • the reaction system of step a) further comprises catalase (EC 1.11.1.6).
  • the catalase is used to remove the by-product hydrogen peroxide because the accumulation of hydrogen peroxide can have a toxic effect on the enzyme catalyst.
  • the catalase can be any enzyme with catalase activity known in the art, such as catalase purchased from Ningxia Xiasheng Industrial Group Co., Ltd. with the commodity code of CAT-400.
  • the reaction of step a) is performed in a reaction buffer.
  • the reaction buffer is a disodium hydrogen phosphate-sodium dihydrogen phosphate buffer at pH 8-9. Better reaction efficiency can be obtained when the reaction is carried out in a disodium hydrogen phosphate-sodium dihydrogen phosphate buffer at pH 8-9.
  • the pH of the transamination reaction of step a) is 6-9, eg, 7-9 or 8-9.
  • step a) the molar ratio of amino acceptor to D,L-glufosinate-ammonium at the start of the reaction is 1:500-1:5.
  • the reaction system of step a) further comprises the coenzyme pyridoxal phosphate.
  • the molar ratio of pyridoxal phosphate to substrate is from 1:10 to 1:200.
  • pyridoxal phosphate is added in an amount of 0.1-2 mM on a molar basis; more preferably 1 mM.
  • the temperature of the transamination reaction in step a) is 25-45°C, such as 30-45°C, 35-45°C, etc.; the time is 10-48 hours, such as 14-48 hours, 24-48 hours , such as 15 hours, 30 hours, etc.
  • step b) the PPO produced in step a) is catalyzed and reduced to L-glufosinate-ammonium by (S)-transaminase, thereby realizing in-situ de-racemization of D,L-glufosinate-ammonium to obtain optically pure L - Glufosinate-ammonium.
  • (S)-transaminase is an enzyme that preferentially induces a transamination reaction of L-glufosinate from racemic D,L-glufosinate in the presence of a ketone substrate such as pyruvate.
  • the (S)-transaminase described herein can be any enzyme known in the art having (S)-transaminase activity.
  • the (S)-transaminase is any one selected from the group consisting of: ATA-0602 (eg, NCBI Serial No. WP_012404126.1), ATA-0607 (eg, NCBI Serial No.
  • the (R)-transaminase is ATA-0611 (EN3).
  • the (S)-transaminase is from Paraburkholderia phymatum, such as ATA-0611 (EN3) from Paraburkholderia phymatum.
  • the amino acid sequence of the (S)-transaminase has at least 70%, 80%, 90%, 91%, 91%, 93%, 94%, 80%, 90%, 91%, 91%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or 100% identity.
  • the nucleotide sequence of the (S)-transaminase is at least 60%, 70%, 80%, 90%, 91%, 91% of the nucleotide sequence shown in SEQ ID No. 8 , 93%, 94%, 95%, 96%, 97%, 98% or 99% or 100% identity.
  • the amino acid sequence of the (S)-transaminase is SEQ ID No.7.
  • the nucleotide sequence of the (S)-transaminase is SEQ ID No. 8.
  • the amino donor in step b) is L-alanine or isopropylamine. In some preferred embodiments, the amino donor is L-alanine.
  • the deamination product of the amino donor in step b) is pyruvate.
  • step b) there is additionally a catalytic enzyme capable of removing the deamination product of the amino donor, in the presence of the catalytic enzyme, the reaction by-product (deamination product of the amino donor, e.g. pyruvate) is removed by the catalytic enzyme to facilitate complete conversion.
  • a catalytic enzyme capable of removing the deamination product of the amino donor, in the presence of the catalytic enzyme, the reaction by-product (deamination product of the amino donor, e.g. pyruvate) is removed by the catalytic enzyme to facilitate complete conversion.
  • the catalytic enzyme capable of removing the deamination product of the amino donor in step b) is pyruvate decarboxylase.
  • the pyruvate decarboxylase (EC 4.1.1.1) described herein may be any enzyme known in the art having pyruvate decarboxylase activity.
  • the pyruvate decarboxylase is Zymobacter palmae.
  • the amino acid sequence of the pyruvate decarboxylase has at least 70%, 80%, 90%, 91%, 91%, 93%, 94%, 95% of the amino acid sequence shown in SEQ ID No. 5 %, 96%, 97%, 98% or 99% or 100% identity.
  • the nucleotide sequence of the pyruvate decarboxylase has at least 60%, 70%, 80%, 90%, 91%, 91%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or 100% identity.
  • the amino acid sequence of the pyruvate decarboxylase is SEQ ID No.5.
  • the nucleotide sequence of the pyruvate decarboxylase is SEQ ID No.6.
  • Enzymes described herein such as (R)-transaminase, (S)-transaminase, catalytic enzymes capable of converting the amino addition product of the amino acceptor to the amino acceptor, enzymes capable of removing the amino donor
  • the catalytic enzyme of the deamination product and the catalase can be in the form of a purified enzyme; partially purified enzyme; cell-free extract or crude cell extract; liquid, powder, or fixed form; permeabilization treatment containing enzymes cells, whole cells or whole fermentation broth or any other suitable form.
  • the catalytic enzyme of the deamination product of the body and the form of said catalase are each independently selected from: partially purified enzyme; cell-free extract or crude cell extract; liquid, powder or fixed form; permeable enzyme-containing Processed cells, whole cells or whole fermentation broth, lyophilized cells, or any combination thereof.
  • the temperature of the transamination reaction in step b) is 25-45°C, such as 30-45°C, 35-45°C, etc.; the time is 10-48 hours, such as 14-48 hours, 24-48 hours , such as 15 hours, 30 hours, etc.
  • the molar ratio of amino donor to substrate at the start of the reaction is 1:2-5:1, such as 1:1, 1.5:1, 2:1, 3:1 or 4:1.
  • the pH of the transamination reaction of step b) is 6-9, eg, 7-9 or 8-9.
  • the reaction of step b) is performed in a reaction buffer.
  • the reaction buffer is a disodium hydrogen phosphate-sodium dihydrogen phosphate buffer at pH 8-9. Better reaction efficiency can be obtained when the reaction is carried out in a disodium hydrogen phosphate-sodium dihydrogen phosphate buffer at pH 8-9.
  • the reaction system of step b) further comprises coenzyme pyridoxal phosphate.
  • the molar ratio of pyridoxal phosphate to substrate is from 1:10 to 1:200.
  • pyridoxal phosphate is added in an amount of 0.1-2 mM on a molar basis; more preferably 1 mM.
  • the present application provides a method for producing L-glufosinate-ammonium, which uses D,L-glufosinate-ammonium as a raw material, and obtains L-glufosinate-ammonium through a multi-enzyme catalytic system.
  • Enzymatic catalytic systems include: (R)-transaminase, D-amino acid oxidase, (S)-transaminase and pyruvate decarboxylase, and optionally catalase.
  • the method includes:
  • step b) transamination of the 2-carbonyl-4-[hydroxy(methyl)phosphono]butyric acid obtained in step a) in the presence of (S)-transaminase, pyruvate decarboxylase and L-alanine The reaction yields L-glufosinate and the resulting pyruvic acid is removed.
  • the reaction mixture obtained in step a) can be thermally treated to deactivate the catalyst, ie, the enzyme used in step a).
  • the heating temperature and time are any suitable temperature and time to inactivate the enzymes in the reaction mixture.
  • the temperature of heating is above 65°C, eg, above 75°C.
  • the heating time is more than 15 minutes, such as 15 minutes to 1 hour, such as 20, 30, 40, 50 minutes.
  • the methods described herein can be carried out in one or more reaction vessels. Preferably, the methods described herein are carried out in one reaction vessel (ie "one pot two step process").
  • the (R)-transaminase used in step a) and the catalytic enzyme eg, D-amino acid oxidase
  • the catalytic enzyme capable of converting the amino addition product of the amino acceptor to the amino acceptor
  • step a) may comprise: in the presence of a first recombinant microorganism and said amino acceptor co-expressing a (R)-transaminase and a catalytic enzyme capable of converting the amino addition product of an amino acceptor to said amino acceptor , the transamination reaction of D,L-glufosinate-ammonium is carried out to obtain 2-carbonyl-4-[hydroxy(methyl)phosphono]butyric acid, and the obtained amino product is converted into the amino acceptor.
  • Utilizing the first recombinant microorganism can impart higher catalytic efficiency to the method of the present application.
  • the first recombinant microorganism can be constructed using any method known in the art.
  • the first recombinant microorganism can be constructed as follows: constructing a recombinant vector containing the (R)-transaminase gene and the catalytic enzyme gene, transforming the recombinant vector into the microorganism, and inducing and culturing the obtained recombinant microorganism, The culture broth is separated to obtain the first recombinant microorganism containing the (R)-transaminase gene and the catalytic enzyme gene.
  • the added amount of the first recombinant microorganism is 5-200 g wet cells/L reaction solution, or, based on the dry cell weight, the first recombinant The added amount of microorganisms is 1-50 g dry cells/L reaction solution.
  • step b) may comprise causing step a) in the presence of a second recombinant microorganism and an amino donor that co-express (S)-transaminase and said catalytic enzyme capable of removing the deamination product of said amino donor.
  • step b) may comprise causing step a) in the presence of a second recombinant microorganism and an amino donor that co-express (S)-transaminase and said catalytic enzyme capable of removing the deamination product of said amino donor.
  • the obtained 2-carbonyl-4-[hydroxy(methyl)phosphono]butyric acid undergoes a transamination reaction to obtain L-glufosinate and removes the obtained reaction by-product, that is, the deamination product of the amino donor.
  • the second recombinant microorganism can confer a higher catalytic efficiency to the method of the present application.
  • the second recombinant microorganism can be constructed using any method known in the art.
  • the second recombinant microorganism can be constructed as follows: constructing a recombinant vector containing the (S)-transaminase and the catalytic enzyme genes, transforming the recombinant vector into the microorganism, inducing and culturing the obtained recombinant microorganism, isolating the The culture broth obtains a second recombinant microorganism containing (S)-transaminase and the catalytic enzyme gene.
  • the added amount of the second recombinant microorganism is 5-200 g wet cells/L reaction solution, or, based on the dry cell weight, the second recombinant microorganism is added in an amount of 5-200 g wet cells/L reaction solution
  • the added amount of microorganisms is 1-50 g dry cells/L reaction solution.
  • the first and second recombinant microorganisms can be any engineered bacteria suitable for enzyme expression.
  • the first and second recombinant microorganisms each independently belong to one of the following genera: Saccharomyces, Aspergillus, Pichia, Kluyveromyces Genus (Kluyveromyces), Candida (Candida), Hansenula (Hansenula), Humicola (Humicola), Issatchenkia (Issatchenkia), Trichosporon (Trichosporon), Brettanomyces ( Brettanomyces), Pachysolen, Yarrowia or Escherichia.
  • the first and second recombinant microorganisms are each independently selected from the group consisting of Saccharomyces cerevisiae, Yarrowia lipolitica, Candida krusei, Issatchenkia orientalis or Escherichia coli. In some more preferred embodiments, both the first and second recombinant microorganisms are E. coli.
  • the yield of the methods of the present application can be measured by any method known in the art.
  • the content of both configurations in the glufosinate product obtained can be measured by chiral HPLC.
  • the glufosinate product is obtained in an enantiomeric excess (e.e.) of at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.9%.
  • SEQ ID NO: 1 is the amino acid sequence annotated as (R)-transaminase (APH1) derived from Pseudarthrobacter chlorophenolicus.
  • SEQ ID NO: 2 is the nucleotide sequence annotated as (R)-transaminase (APH1) derived from Pseudarthrobacter chlorophenolicus.
  • SEQ ID NO: 3 is the amino acid sequence annotated as D-amino acid oxidase (DAAO) derived from Rhodotorula sp. CCFEE 5036.
  • DAAO D-amino acid oxidase
  • SEQ ID NO: 4 is the nucleotide sequence annotated as D-amino acid oxidase (DAAO) derived from Rhodotorula sp. CCFEE 5036.
  • DAAO D-amino acid oxidase
  • SEQ ID NO: 5 is the amino acid sequence derived from Zymobacter palmae annotated as pyruvate decarboxylase (PDC).
  • SEQ ID NO:6 is the nucleotide sequence derived from Zymobacter palmae annotated as pyruvate decarboxylase (PDC).
  • SEQ ID NO: 7 is the amino acid sequence derived from Paraburkholderia phymatum STM815 annotated as (S)-transaminase (EN3).
  • SEQ ID NO: 8 is the nucleotide sequence derived from Paraburkholderia phymatum STM815 annotated as (S)-transaminase (EN3).
  • Fig. 1 is the reaction formula for producing L-glufosinate-ammonium by the multi-enzyme system splitting method adopted in some embodiments of the application.
  • Figure 2-1 exemplifies the use of recombinant E. coli BL21(DE3)/pET-28a-APH1 single enzyme and E.coli BL21(DE3)/pET-28a-APH1&E.coli BL21(DE3)/pET- 28a-DAAO catalytic efficiency comparison bar graph
  • Figure 2-2 exemplarily shows E.coli BL21(DE3)/pET-28a-APH1 and E.coli BL21(DE3)/pET-28a-DAAO resolution racemic PPT Reaction progress curve.
  • Figure 3 exemplarily shows the reaction progress curves for the synthesis of L-PPT by reductive amination of PPO with recombinant E. coli BL21(DE3)/pET-28a-EN3 and E. coli BL21(DE3)/pET-28a-PDC.
  • Figure 4 exemplarily shows the APH1-DAAO construct.
  • Figure 5 exemplarily shows the EN3-PDC construct.
  • Figure 6 exemplarily shows a reaction progress curve for the resolution of racemic PPT with recombinant E. coli co-expression strain E. coli BL21(DE3)/pCDFduet-1-APH1-DAAO.
  • Figure 7 exemplarily shows the reaction progress curve for the synthesis of L-PPT by reductive amination of PPO with recombinant E. coli co-expression strain E. coli BL21(DE3)/pCDFduet-1-EN3-PDC.
  • FIG. 8 exemplarily shows the reaction progress curve of preparation of L-glufosinate-ammonium by double bacteria multi-enzyme one-pot two-step deracemization.
  • genome extraction kit plasmid extraction kit, DNA purification and recovery kit
  • One-step cloning kit was obtained from Vazyme Co., Ltd. MultiS One Step Cloning Kit
  • E.coli DH5 ⁇ , E.coli BL21(DE3), plasmid pET-28a(+), pCDFduet-1 vector, etc. were purchased from Shanghai Xuguan Biotechnology Development Co., Ltd.
  • DNA marker, low molecular weight standard protein , protein glue, etc. were purchased from Beijing GenStar Co., Ltd.
  • catalase was purchased from Ningxia Xiasheng Industrial Group Co., Ltd., the commodity number is CAT-400. Refer to the product manual for the usage of the above reagents.
  • the reagents used in the catalytic process 2-carbonyl-4-[hydroxy(methyl)phosphono]butyric acid (PPO for short), D,L-glufosinate-ammonium were purchased from Yongnong Bioscience Co., Ltd.; other commonly used reagents were purchased from Sinopharm Group Chemical Reagent Co., Ltd.
  • HPLC analysis method is: chromatographic column/PBr; column temperature/30°C; flow rate/0.8mL/min; detection wavelength/210nm; mobile phase: 50mM (NH4)2HPO4, adding 12% acetonitrile.
  • the content of two configurations of glufosinate-ammonium was checked by chiral HPLC analysis method.
  • the chiral HPLC analysis method was: chromatographic column/OA-5000L; mobile phase/0.5% copper sulfate pentahydrate; detection wavelength/254nm; flow rate/0.7mL /min; column temperature/35°C.
  • the engineered bacteria were recombined with E.coli BL21(DE3)/pET-28a-APH1, E.coli BL21(DE3)/pET-28a-DAAO, E.coli BL21(DE3)/pET-28a-EN3, E.coli BL21(DE3)/pET-28a-DAAO, E. After the coli BL21(DE3)/pET-28a-PDC was activated by streaking on a plate, a single colony was inoculated into 10 mL of LB liquid medium containing 50 ⁇ g/mL kanamycin, and incubated at 37 °C with shaking for 10 h.
  • APH1 The sequence derived from Pseudarthrobacter chlorophenolicus annotated as (R)-transaminase (APH1) (the amino acid sequence is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO.2) was synthesized after the whole gene was inserted.
  • the plasmid pET-28a(+) was expressed to give pET28a-APH1.
  • pET28a-APH1 was transferred into the expression host E. coli BL21 (DE3) for subsequent expression of the recombinase.
  • the sequence (amino acid sequence shown in SEQ ID NO.3, nucleotide sequence shown in SEQ ID NO.4) annotated as D-amino acid oxidase (DAAO) derived from Rhodotorula sp.CCFEE 5036 was subjected to full gene synthesis Then, insert the expression plasmid pET-28a(+) to obtain pET28a-DAAO. After sequencing and verification, the pET28a-DAAO was transferred into the expression host E.coli BL21(DE3) for subsequent expression of the recombinase.
  • DAAO D-amino acid oxidase
  • the sequence derived from Zymobacter palmae annotated as pyruvate decarboxylase (PDC) (the amino acid sequence is shown in SEQ ID NO.5, and the nucleotide sequence is shown in SEQ ID NO.6) is fully synthesized and inserted into the expression plasmid pET-28a(+) yields pET28a-PDC. After sequencing and verification, the pET28a-PDC was transferred into the expression host E. coli BL21 (DE3) for subsequent recombinase expression.
  • PDC pyruvate decarboxylase
  • Example 3 Resolution of racemic PPT with recombinant E. coli BL21(DE3)/pET-28a-APH1 and E. coli BL21(DE3)/pET-28a-DAAO
  • D-PPT split process curve experiment 30ml reaction system contains 500mM D,L-PPT, 10mM pyruvate, 4000U/mL catalase and 100mM phosphate buffer, the pH of the reaction system is adjusted with ammonia water Adjust to 8.0, and add 30g/L stem cells of recombinant E.coli BL21(DE3)/pET-28a-APH1 and E.coli BL21(DE3)/pET-28a-DAAO.
  • the reaction conditions were as follows: temperature at 30° C. and rotation speed at 250 rpm. Samples (100 ⁇ l) were taken at regular intervals, 900 ⁇ l of deionized water was added, and the reaction was terminated by heating. The conversion of D-PPT was detected by HPLC, and the reaction progress curve was shown in Figure 2-2.
  • Example 4 Synthesis of L-PPT by reductive amination of PPO with recombinant E. coli BL21(DE3)/pET-28a-EN3 and E. coli BL21(DE3)/pET-28a-PDC
  • the 30ml reaction system contains 500mM PPO, 1.2M L-alanine, 100mM phosphate buffer, the pH of the reaction system is adjusted to 8.0 with ammonia, and recombinant Escherichia coli E.coli BL21(DE3)/pET-28a- EN3 and pET-28a-PDC stem cells at 30 g/L each.
  • the reaction conditions were as follows: temperature at 30° C. and rotation speed at 250 rpm. Samples (100 ⁇ l) were taken at regular intervals, 900 ⁇ l of deionized water was added, and the reaction was terminated by heating. The conversion of PPO was detected by HPLC, and the reaction progress curve was shown in FIG. 3 .
  • the APH1 gene sequence used in Example 2 was connected to the multi-cloning site vector pCDFduet-1 by a one-step cloning kit, the restriction sites were HindIII and XhoI, and the one-step cloning primers were C1-F and C1-R (Table 1). ) to construct the plasmid pCDFduet-1-APH1.
  • the DAAO fragment used in Example 2 was connected to the second cloning site of the multi-cloning site vector pCDFduet-1 through the one-step cloning kit.
  • the one-step cloning primers were C2-F and C2-R (Table 1), the plasmid pCDFduet-1-APH1-DAAO was constructed and the plasmid was transformed into strain E.coli BL21 (DE3) to obtain co-expression strain E. coli BL21(DE3)/pCDFduet-1-APH1-DAAO.
  • Figure 4 shows the APH1-DAAO construct.
  • the EN3 gene used in Example 2 was connected to the multi-cloning site vector pCDFduet-1 by a one-step cloning kit, the restriction sites were NcoI and HindIII, and the one-step cloning primers were C3-F and C3-R (Table 1) , the plasmid pCDFduet-1-EN3 was constructed.
  • the PDC was connected to the second cloning site of the multi-cloning site vector pCDFduet-1 through the one-step cloning kit, and the restriction sites were NdeI and EcoRI, and one-step cloning was performed.
  • the primers were C4-F and C4-R (Table 1), the plasmid pCDFduet-1-EN3-PDC was constructed and the plasmid was transferred into the strain E.coli BL21(DE3) to obtain the co-expression strain E.coli BL21(DE3)/pCDFduet -1-EN3-PDC.
  • Figure 5 shows the EN3-PDC construct.
  • Example 6 Resolution of racemic PPT with recombinant E. coli co-expression strain E. coli BL21(DE3)/pCDFduet-1-APH1-DAAO
  • the co-expression strain E.coli BL21(DE3)/pCDFduet-1-APH1-DAAO capable of expressing (R)-transaminase and D-amino acid oxidase was constructed and cultured according to the method of Example 5(1).
  • the 30ml reaction system contains 500mM D,L-PPT, 10mM pyruvate, 4000U/mL catalase, 100mM phosphate buffer, the pH of the reaction system is adjusted to 8.0 with ammonia, and the co-expression strain E.coli BL21 is added. (DE3)/pCDFduet-1-APH1-DAAO 30g/L stem cells.
  • the reaction conditions were as follows: temperature at 30° C. and rotation speed at 250 rpm. Samples (100 ⁇ l) were taken at regular intervals, 900 ⁇ l of deionized water was added, and the reaction was terminated by heating. The conversion of D-PPT was detected by HPLC, and the reaction progress curve was shown in FIG. 6 .
  • Example 7 Synthesis of L-PPT by reductive amination of PPO with co-expression strain E. coli BL21(DE3)/pCDFduet-1-EN3-PDC
  • the co-expression strain E.coli BL21(DE3)/pETduet-1-EN3-PDC capable of expressing (S)-transaminase and pyruvate decarboxylase was constructed and cultured according to the method of Example 5(2), and the bacterial cells were collected by centrifugation.
  • the 30ml reaction system contains 500mM PPO, 1.2M L-alanine, 100mM phosphate buffer, the pH of the reaction system is adjusted to 8.0 with ammonia, and the co-expression strain E.coli BL21(DE3)/pCDFduet-1-EN3 is added -PDC 30g/L stem cells.
  • the reaction conditions were as follows: temperature at 30° C. and rotation speed at 250 rpm. Samples (100 ⁇ l) were taken at regular intervals, 900 ⁇ l of deionized water was added, and the reaction was terminated by heating. The conversion of PPO was detected by HPLC, and the reaction progress curve was shown in FIG. 7 .
  • the co-expression strain E.coli BL21(DE3)/pCDFduet-1-APH1-DAAO capable of expressing (R)-transaminase and D-amino acid oxidase was constructed and cultured according to the method of Example 5(1).
  • the co-expression strain E.coli BL21(DE3)/pETduet-1-EN3-PDC capable of expressing (S)-transaminase and pyruvate decarboxylase was constructed and cultured according to the method of Example 5(2), and the bacterial cells were collected by centrifugation.

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Abstract

本申请涉及一种利用生物多酶偶联法制备L-草铵膦的方法，其包括：a)在(R)-转氨酶和氨基受体的存在下，使D,L-草铵膦发生转氨反应得到2-羰基-4-[羟基(甲基)膦酰基]丁酸和氨基受体的加氨基产物；b)在(S)-转氨酶和氨基供体的存在下，使步骤a)中得到的2-羰基-4-[羟基(甲基)膦酰基]丁酸发生转氨反应得到L-草铵膦和氨基供体的脱氨基产物。本申请的方法能够实现高浓度D,L-草铵膦的高效拆分来制备L-草铵膦。

Description

利用生物多酶偶联法制备L-草铵膦的方法
相关申请的交叉引用
本申请要求享有2020年12月17日提交的名称为“利用生物多酶偶联法制备L-草铵膦的方法”的中国专利申请第202011491692.6号的优先权，并且该专利申请以引用的方式并入本文中。
技术领域
本申请涉及生物技术领域，特别是涉及一种利用生物多酶偶联法制备L-草铵膦的方法。
背景技术
草铵膦(又名双丙氨膦、草丁膦，商品名包括保试达、百速顿等，英文名为phosphinothricin(简称PPT)，化学名为2-氨基-4-[羟基(甲基)膦酰基]丁酸)是德国赫斯特公司(现属于拜耳公司)在20世纪80年代开发的一种具有低毒、高效、非选择性触杀型有机磷除草剂。草铵膦作用机制是，草铵膦作用植株后，可以抑制谷氨酰胺合成酶，从而中断植物体内谷氨酸的可逆反应，造成代谢紊乱，堆积过量的氨，使植物中毒，同时导致植物无法合成叶绿素，叶绿体解体，植物的光合作用就会受到抑制，导致植物死亡。草铵膦主要用于果园、马铃薯田、非耕地等防治一年生和多年生的禾本科及双子叶杂草，如马唐、狗尾草、野小麦；多年生的禾本科杂草和莎草，如羊茅、鸭芽等。
草铵膦有两种光学异构体，分别为L-草铵膦和D-草铵膦，但只有L-型具有除草活性，且在土壤中易分解，对人类和动物的毒性较小，除草谱广，对环境的破坏力小。
目前，市场上销售的草铵膦一般都是外消旋混合物。若草铵膦产品能以L-构型的纯光学异构体形式使用，可显著降低草铵膦的使用量，这对于提高原子经济性、降低使用成本、减轻环境压力具有重要意义。
现有制备手性纯L-草铵膦的方法主要两种：化学法和生物法。
其中化学法包括化学拆分法和化学合成法。
化学拆分法是通过手性拆分试剂拆分外化学法合成的消旋D,L-草铵膦或其衍生物，从而制得光学纯的L-草铵膦。Hoechst公司于1998年报道了利用奎宁作为拆分剂拆分草铵膦消旋体，实现了D-和L-草铵膦的分离(美国专利US5767309)。然而该工艺存在需要使用手性拆分试剂、D-草铵膦需要消旋再利用，需要多次拆分等缺点，难以规模工业化生产。
化学合成法以天然手性氨基酸或者不对称法合成L-草铵膦。例如Hoechst公司报道了利用L-谷氨酸或L-天冬氨酸为手性源合成L-草铵膦(Hoffmann M G,Zeiss H J.A novel and convenient route to L-homoserine lactones and L-phosphinothricin from L-aspartic acid[J].Tetrahedron Letters,1992,33(19):2669-2672.和欧洲专利EP0530506)。但是该过程氨基保护与脱保护过程复杂，步骤较多，多见于实验室研究中。L-草铵膦工业化是明治制果采用不对称催化加氢法率先实现的(国际专利WO 2006104120)。但该路线所使用的催化剂价格昂贵，反应路线较长，工业化成本较高。
相比之下，生物催化法具有反应条件温和、立体选择性高、收率高等优点，是工业化制备L-草铵膦的重要趋势。
生物催化法制备L-草铵膦的方法目前大多是2-氧代-4-(羟基甲基氧膦基)丁酸(PPO)为底物，经氨化反应，不对称合成L-草铵膦。其中CN1349561A描述了将天冬氨酸作为氨基供体，利用筛选的天冬氨酸转氨酶(Asp-TA)通过转氨作用将PPO合成L-草铵膦。该工艺使用与PPO大约等摩尔量的氨基供体，生成的草酰乙酸在水中水解成丙酮酸，经酶促反应去除，不存在可逆反应。然而此工艺效率较低，底物PPO转化率仅有52％，反应条件苛刻(80℃)。专利CN105603015B描述了一种以L-丙氨酸为氨基供体的氨基转移酶生产L-草铵膦的方法。该工艺能够将100mM的PPO完全转化成L-草铵膦，但是为了抑制逆反应，丙氨酸投量过高，反应结束仍有300mM的L-丙氨酸存在体系中，为后续分离纯化造成困难，同时反应温度高达50℃，能耗较高，不利于生产。另外还有的方法在氨基酸脱氢酶的介导下，以无机氨为供体，将PPO还原胺化制备L-精草铵膦，例如CN106978453A，反应过程采用无机氨，转化率能够达到99％以上，然而该工艺需要额外添加辅酶再生系统，提高了分离纯化的难度。
上述工艺都以PPO为原料，但PPO的成本很高，从而导致这些工艺生产L-草铵膦的成本很高，难以实现工业化。因此，国际专利WO2017151573A1描述了一种以D，L-草铵膦为原料生产L-精草铵膦的方法，该工艺采用D-氨基酸氧化酶氧化D-草铵膦获得PPO，再由转氨酶经转氨反应将PPO转化为L-草铵膦。尽管该工艺实现了草铵膦消旋体的动态动力学拆分，但是该工艺存在明显缺陷：一是底物投量难以提升(仅有300mM D,L-草铵膦)；二是转氨酶介导的PPO到L-草铵膦的反应，由于受到可逆反应影响，仅能实现90％的转化率；三是由于胺供体是L-谷氨酸，反应后仍有大量剩余，产物分离纯化困难。
因此迫切需要开发一种能够高效实现动态拆分高浓度D,L-草铵膦来制备L-草铵膦的方法。
发明内容
本申请提供了一种利用生物多酶偶联法氧化还原不对称制备L-草铵膦的方法，其以D,L-草铵膦为原料，经多酶催化体系获得L-草铵膦。该方法原料转化率高、分离精制过程简单、产品收率高、生产成本低，易于工业化。
在一些实施方式中，本申请提供了一种制备L-草铵膦的方法，其包括：
a)在(R)-转氨酶和氨基受体的存在下，使D,L-草铵膦发生转氨反应得到2-羰基-4-[羟基(甲基)膦酰基]丁酸和氨基受体的加氨基产物；
b)在(S)-转氨酶和氨基供体的存在下，使步骤a)中得到的2-羰基-4-[羟基(甲基)膦酰基]丁酸发生转氨反应得到L-草铵膦和氨基供体的脱氨基产物。
在步骤a)中，以外消旋D,L-草铵膦为底物，在微量氨基受体例如丙酮酸(反应后无需分离)存在的条件下，(R)-转氨酶将D-草铵膦的氨基转移到氨基受体上，脱氨生成2-羰基-4-(羟基甲基膦酰基)丁酸(PPO)，而L-草铵膦不参与反应而完全保留。
转氨酶(Amine Transaminase,ATA,EC 2.6.1.X)属于转移酶类，是催化1个氨基供体(氨基酸或简单的胺)上的氨基转移到前手性的受体酮，得到手性胺和副产物酮或者α-酮酸的一类酶，其催化的反应是可逆的。根据氨基被转移到不同位置的氨基受体上，转氨酶又可以被分为α-转氨酶和ω-转氨酶(EC2.6.1.1)。ω-转氨酶反应过程可分为两步，第一步反应是在ω-转氨酶的作用下将氨基供体上的氨基转移到PLP的羰基上，从而形成5-磷酸吡哆胺(PMP)和 与氨基供体对应的酮；第二步反应同样在ω-转氨酶的作用下将PMP上的氨基转移到氨基受体上，PMP又转变为PLP实现循环。转氨酶的立体选择性可在包含手性中心的底物外消旋混合物的外消旋拆分中测定。ω-转氨酶根据立体选择性可分为“(R)-转氨酶”((R)-amine transaminase)和(S)-转氨酶((S)-amine transaminase)。
根据本申请，(R)-转氨酶可以是在存在酮底物(例如丙酮酸)的情况下，优先从外消旋D,L-草铵膦中诱导D-草铵膦的转氨反应的酶。(R)-转氨酶可以为本领域已知的任何具有(R)-转氨酶活性的酶。在一些实施方式中，所述(R)-转氨酶为选自以下中的任一种：APH1(例如NCBI序列号为WP_015938787.1)、HEA-2(例如NCBI序列号为ABX05998.1)、TSP-1(例如NCBI序列号为WP_013128145.1)、DEP-2(例如NCBI序列号为WP_013615256.1)和MPH(例如NCBI序列号为WP_013863226.1)。在一些实施方式中，所述(R)-转氨酶为APH1。在一些实施方式中，所述(R)-转氨酶来自Pseudarthrobacter chlorophenolicus，例如来自Pseudarthrobacter chlorophenolicus的APH1。在一些实施方式中，所述(R)-转氨酶的氨基酸序列与SEQ ID No.1所示的氨基酸序列具有至少70％、80％、90％、91％、91％、93％、94％、95％、96％、97％、98％或99％或100％的同一性。在一些实施方式中，所述(R)-转氨酶的核苷酸序列与SEQ ID No.2所示的核苷酸序列具有至少60％、70％、80％、90％、91％、91％、93％、94％、95％、96％、97％、98％或99％或100％的同一性。在一些实施方式中，所述(R)-转氨酶的氨基酸序列为SEQ ID No.1。在一些实施方式中，所述(R)-转氨酶的核苷酸序列为SEQ ID No.2。
在一些实施方式中，步骤a)中的所述氨基受体为丙酮酸、丙酮酸甲酯或丙酮酸乙酯。在一些优选的实施方式中，步骤a)中的所述氨基受体为丙酮酸。
在一些实施方式中，步骤a)中所述氨基受体的加氨基产物为D-丙氨酸。
在一些实施方式中，步骤a)中另外存在能够使所述氨基受体的加氨基产物转化为所述氨基受体的催化酶。这样，氨基受体得到氨基转化为的加氨基产物在所述催化酶的作用下又转化回氨基受体，从而在使反应体系具有更好的催化效率的同时实现氨基受体的原位再生，避免了大量氨基受体的使用。
在一些实施方式中，步骤a)中能够使所述氨基受体的加氨基产物转化为所述氨基受体的所述催化酶为D-氨基酸氧化酶，其使得D-丙氨酸转化为丙酮酸。
本申请所述的D-氨基酸氧化酶(EC 1.4.3.3)可以为本领域已知的任何具有D-氨基酸氧化酶活性的酶。在一些实施方式中，所述D-氨基酸氧化酶来自Rhodotorula sp.。在一些实施方式中，所述D-氨基酸氧化酶来自Rhodotorula sp.CCFEE 5036。在一些实施方式中，所述D-氨基酸氧化酶的氨基酸序列与SEQ ID No.3所示的氨基酸序列具有至少70％、80％、90％、91％、91％、93％、94％、95％、96％、97％、98％或99％或100％的同一性。在一些实施方式中，所述D-氨基酸氧化酶的核苷酸序列与SEQ ID No.4所示的核苷酸序列具有至少60％、70％、80％、90％、91％、91％、93％、94％、95％、96％、97％、98％或99％或100％的同一性。在一些实施方式中，所述D-氨基酸氧化酶的氨基酸序列为SEQ ID No.3。在一些实施方式中，所述D-氨基酸氧化酶的核苷酸序列为SEQ ID No.4。
在一些实施方式中，步骤a)的反应体系中还包括过氧化氢酶(EC1.11.1.6)。所述过氧化氢酶用于去除副产物过氧化氢，因为过氧化氢积累会对酶催化剂有毒害作用。过氧化氢酶可以为本领域已知的任何具有过氧化氢酶活性的酶，例如购自宁夏夏盛实业集团有限公司，商品编号为CAT-400的过氧化氢酶。
在一些实施方式中，步骤a)的反应在反应缓冲液中进行。优选地，所述反应缓冲液是pH为8-9的磷酸氢二钠-磷酸二氢钠缓冲液。在pH为8-9的磷酸氢二钠-磷酸二氢钠缓冲液中进行反应时可以获得更优的反应效率。
在一些实施方式中，步骤a)的转氨反应的pH值为6-9，例如7-9或8-9。
在一些实施方式中，在步骤a)中，反应开始时氨基受体与D,L-草铵膦的摩尔比为1:500-1:5。
在一些实施方式中，步骤a)的反应体系中还包括辅酶磷酸吡哆醛。在一些实施方式中，磷酸吡哆醛与底物的摩尔比为1:10-1:200。在一些实施方式中，以摩尔浓度计，磷酸吡哆醛的添加量为0.1-2mM；更优选为1mM。
在一些实施方式中，步骤a)的转氨反应的温度为25-45℃，例如30-45℃，35-45℃等；时间为10-48小时，例如14-48小时，24-48小时，例如15小时、30小时等。
在步骤b)中，步骤a)中产生的PPO被(S)-转氨酶催化还原为L-草铵膦，从而实现D,L-草铵膦的原位去消旋化，得到光学纯的L-草铵膦。
根据本发明，(S)-转氨酶是在存在酮底物比如丙酮酸的情况下，优先从外消旋D,L-草铵膦诱导L-草铵膦的转氨反应的酶。本申请所述的(S)-转氨酶可以为本领域已知的任何具有(S)-转氨酶活性的酶。在一些实施方式中，所述(S)-转氨酶为选自以下中的任一种：ATA-0602(例如NCBI序列号为WP_012404126.1)、ATA-0607(例如NCBI序列号为WP_012404467.1)、ATA-0611(EN3)(例如NCBI序列号为WP_012403900.1)、ATA-0701(例如NCBI序列号为WP_013601929.1)和ATA-0801(例如NCBI序列号为WP_013614910.1)。在一些实施方式中，所述(R)-转氨酶为ATA-0611(EN3)。在一些实施方式中，所述(S)-转氨酶来自Paraburkholderia phymatum，例如来自Paraburkholderia phymatum的ATA-0611(EN3)。在一些实施方式中，所述(S)-转氨酶的氨基酸序列与SEQ ID No.7所示的氨基酸序列具有至少70％、80％、90％、91％、91％、93％、94％、95％、96％、97％、98％或99％或100％的同一性。在一些实施方式中，所述(S)-转氨酶的核苷酸序列与SEQ ID No.8所示的核苷酸序列具有至少60％、70％、80％、90％、91％、91％、93％、94％、95％、96％、97％、98％或99％或100％的同一性。在一些实施方式中，所述(S)-转氨酶的氨基酸序列为SEQ ID No.7。在一些实施方式中，所述(S)-转氨酶的核苷酸序列为SEQ ID No.8。
在一些实施方式中，步骤b)中的所述氨基供体为L-丙氨酸或异丙胺。在一些优选的实施方式中，所述氨基供体为L-丙氨酸。
在一些实施方式中，步骤b)中的所述氨基供体的脱氨基产物为丙酮酸。
在一些实施方式中，步骤b)中另外存在能够除去所述氨基供体的脱氨基产物的催化酶，在该催化酶的存在下，所述反应副产物(氨基供体的脱氨基产物，例如丙酮酸)通过所述催化酶被除去以利于完全转化。
在一些实施方式中，步骤b)中能够除去所述氨基供体的脱氨基产物的所述催化酶为丙酮酸脱羧酶。
本申请所述的丙酮酸脱羧酶(EC 4.1.1.1)可以为本领域已知的任何具有丙酮酸脱羧酶活性的酶。在一些实施方式中，所述丙酮酸脱羧酶是Zymobacter palmae。在一些实施方式中，所述丙酮酸脱羧酶的氨基酸序列与SEQ ID No.5所示的氨基酸序列具有至少70％、80％、90％、91％、91％、93％、94％、95％、96％、97％、98％或99％或100％的同一性。在一些实施方式中，所述丙酮酸脱羧酶的核苷酸序列与SEQ ID No.6所示的核苷酸序列具有至少60％、70％、80％、90％、91％、91％、93％、94％、95％、96％、97％、98％或99％或100％的同一性。在一些实施方式中，所述丙酮酸脱羧酶的氨基酸序列为SEQ ID No.5。在一些实施方式中，所述丙酮酸脱羧酶的核苷酸序列为SEQ ID No.6。
本申请所述的酶，例如(R)-转氨酶、(S)-转氨酶、能够使所述氨基受体的加氨基产物转化为所述氨基受体的催化酶、能够除去所述氨基供体的脱氨基产物的催化酶和所述过氧化氢酶的形式可以为纯化的酶；部分纯化的酶；无细胞提取物或粗细胞提取物；液体、粉末或固定形式；含有酶的可透化处理的细胞、完整细胞或完整发酵液或其他任何合适形式。因此，在一些实施方式中，所述(R)-转氨酶、(S)-转氨酶、能够使所述氨基受体的加氨基产物转化为所述氨基受体的催化酶、能够除去所述氨基供体的脱氨基产物的催化酶和所述过氧化氢酶的形式各自独立地选自：部分纯化的酶；无细胞提取物或粗细胞提取物；液体、粉末或固定形式；含有酶的可透化处理的细胞、完整细胞或完整发酵液、冻干细胞或其任何组合。
在一些实施方式中，步骤b)的转氨反应的温度为25-45℃，例如30-45℃，35-45℃等；时间为10-48小时，例如14-48小时，24-48小时，例如15小时、30小时等。
在一些实施方式中，在步骤b)中，反应开始时氨基供体与底物的摩尔比为1:2-5:1，例如1:1、1.5:1、2:1、3:1或4:1。
在一些实施方式中，步骤b)的转氨反应的pH值为6-9，例如7-9或8-9。
在一些实施方式中，步骤b)的反应在反应缓冲液中进行。优选地，所述反应缓冲液是pH为8-9的磷酸氢二钠-磷酸二氢钠缓冲液。在pH为8-9的磷酸氢二钠-磷酸二氢钠缓冲液中进行反应时可以获得更优的反应效率。
在一些实施方式中，步骤b)的反应体系中还包括辅酶磷酸吡哆醛。在一些实施方式中，磷酸吡哆醛与底物的摩尔比为1:10-1:200。在一些实施方式中，以摩尔浓度计，磷酸吡哆醛的添加量为0.1-2mM；更优选为1mM。
在一些优选的实施方式中，本申请提供了一种生产L-草铵膦的方法，其以D,L-草铵膦为原料，经多酶催化体系获得L-草铵膦，所述多酶催化体系包括：(R)-转氨酶、D-氨基酸氧化酶、(S)-转氨酶和丙酮酸脱羧酶，以及任选地过氧化氢酶。所述方法包括：
a)在(R)-转氨酶、D-氨基酸氧化酶和丙酮酸的存在下，使D,L-草铵膦发生转氨反应得到2-羰基-4-[羟基(甲基)膦酰基]丁酸并使得到的D-丙氨酸转化为丙酮酸；
b)在(S)-转氨酶、丙酮酸脱羧酶和L-丙氨酸的存在下，使步骤a)中获得的2-羰基-4-[羟基(甲基)膦酰基]丁酸发生转氨反应得到L-草铵膦并除去得到的丙酮酸。
在一些实施方式中，步骤a)的反应完成后和进行步骤b)之前，可以对步骤a)得到的反应混合物进行热处理，从而灭活催化剂，即步骤a)中所用的酶。加热温度和时间为使反应混合物中的酶失活的任何合适温度和时间。在一些实施方式中，加热的温度为65℃以上，例如75℃以上。在一些实施方式中，加热的时间为15分钟以上，例如15分钟至1小时，例如20、30、40、50分钟。
本申请所述的方法可以在一个或更多个反应容器中进行。优选地，本申请所述的方法在一个反应容器中进行(即“一锅两步法”)。
在一些优选的实施方式中，步骤a)中使用的(R)-转氨酶和能够使所述氨基受体的加氨基产物转化为所述氨基受体的催化酶(例如D-氨基酸氧化酶)由第一重组微生物共表达。因此，步骤a)可以包括：在共表达(R)-转氨酶和能够使氨基受体的加氨基产物转化为所述氨基受体的催化酶的第一重组微生物和所述氨基受体的存在下，使D,L-草铵膦发生转氨反应得到2-羰基-4-[羟基(甲基)膦酰基]丁酸并使得到的所述加氨基产物转化为所述氨基受体。利用所述第一重组微生物能够赋予本申请方法更高的催化效率。可以利用本领域已知的任何方法构建所述第一重组微生物。例如，所述第一重组微生物可以如下构建：构建含所述(R)-转氨酶基因和所述催化酶基因的重组载体，将所述重组载体转化至微生物，对获得的重组微生物进行诱导培养，分离培养液得到含有(R)-转氨酶基因 和所述催化酶基因的第一重组微生物。优选地，按照10000rpm离心10min后的菌体湿重计，所述第一重组微生物的添加量为5-200g湿菌体/L反应液，或者，以干菌体重量计，所述第一重组微生物的添加量为1-50g干菌体/L反应液。
在一些优选的实施方式中，步骤b)中使用的(S)-转氨酶和所述能够除去所述氨基供体的脱氨基产物的催化酶由第二重组微生物共表达。因此，步骤b)可以包括：在共表达(S)-转氨酶和所述能够除去所述氨基供体的脱氨基产物的催化酶的第二重组微生物和氨基供体的存在下，使步骤a)获得的2-羰基-4-[羟基(甲基)膦酰基]丁酸发生转氨反应得到L-草铵膦并除去得到的反应副产物，即所述氨基供体的脱氨基产物。利用所述第二重组微生物能够赋予本申请方法更高的催化效率。可以利用本领域已知的任何方法构建所述第二重组微生物。例如，所述第二重组微生物可以如下构建：构建含所述(S)-转氨酶和所述催化酶基因的重组载体，将所述重组载体转化至微生物，对获得的重组微生物进行诱导培养，分离培养液得到含有(S)-转氨酶和所述催化酶基因的第二重组微生物。优选地，按照10000rpm离心10min后的菌体湿重计，所述第二重组微生物的添加量为5-200g湿菌体/L反应液，或者，以干菌体重量计，所述第二重组微生物的添加量为1-50g干菌体/L反应液。
所述第一和第二重组微生物可以是任何适用于酶表达的工程菌。在一些实施方式中，所述第一和第二重组微生物各自独立地属于以下属中的一种：酵母属(Saccharomyces)、曲霉属(Aspergillus)、毕赤酵母属(Pichia)、克鲁维酵母属(Kluyveromyces)、假丝酵母属(Candida)、汉逊酵母属(Hansenula)、腐质霉属(Humicola)、伊萨酵母属(Issatchenkia)、毛孢子菌属(Trichosporon)、酒香酵母属(Brettanomyces)、管囊酵母属(Pachysolen)、耶氏酵母属(Yarrowia)或埃希氏杆菌属(Escherichia)。在一些优选的实施方式中，所述第一和第二重组微生物各自独立地选自酿酒酵母(Saccharomyces cerevisiae)、解脂耶氏酵母(Yarrowia lipolitica)、克鲁斯假丝酵母(Candida krusei)、东方伊萨酵母(Issatchenkia orientalis)或大肠杆菌(Escherichia coli)。在一些更优选的实施方式中，所述第一和第二重组微生物均是大肠杆菌。
本申请方法的产率可以通过本领域已知的任何方法测量。例如，可以通过手性HPLC来测量所获得的草铵膦产物中两个构型含量。在一些实施方式中， 获得的草铵膦产物的对映体过量(e.e.)至少为80％、85％、90％、95％、96％、97％、98％、99％或99.9％。
本申请方法具有以下有益效果：
(1)D-氨基酸氧化酶的引入使反应体系具有更好的催化效率，以外消旋D,L-草铵膦为底物进行催化反应时，转化率远高于单酶催化，PPO产率也大幅提升。
(2)D-草铵膦被氧化为2-羰基-4-[羟基(甲基)膦酰基]丁酸，L-草铵膦因不参与反应而被完全保留；产物2-羰基-4-[羟基(甲基)膦酰基]丁酸又可以被转氨酶继续催化还原为L-草铵膦，进而实现D,L-草铵膦的原位去消旋化。而传统的氧化方法则需要将D-草铵膦和L-草铵膦都转化为2-羰基-4-[羟基(甲基)膦酰基]丁酸，造成了原料的浪费。
(3)能够直接以D,L-草铵膦为底物进行拆分，无需昂贵的拆分试剂，也无需合成草铵膦衍生物，更无需对D-草铵膦进行分离、再消旋、再拆分等步骤。
(4)克服了化学法合成L-草铵膦前体2-羰基-4-[羟基(甲基)膦酰基]丁酸的缺陷，是一种绿色，环保，低碳的工艺路线，适合大规模工业化生产应用。
对序列表的描述
SEQ ID NO:1是来源于Pseudarthrobacter chlorophenolicus的注释为(R)-转氨酶(APH1)的氨基酸序列。
SEQ ID NO:2是来源于Pseudarthrobacter chlorophenolicus的注释为(R)-转氨酶(APH1)的核苷酸序列。
SEQ ID NO:3是来源于Rhodotorula sp.CCFEE 5036的注释为D-氨基酸氧化酶(DAAO)的氨基酸序列。
SEQ ID NO:4是来源于Rhodotorula sp.CCFEE 5036的注释为D-氨基酸氧化酶(DAAO)的核苷酸序列。
SEQ ID NO:5是来源于Zymobacter palmae注释为丙酮酸脱羧酶(PDC)的氨基酸序列。
SEQ ID NO:6是来源于Zymobacter palmae注释为丙酮酸脱羧酶(PDC)的核苷酸序列。
SEQ ID NO:7是来源于Paraburkholderia phymatum STM815注释为(S)-转氨酶(EN3)的氨基酸序列。
SEQ ID NO:8是来源于Paraburkholderia phymatum STM815注释为(S)-转氨酶(EN3)的核苷酸序列。
附图说明
图1为本申请的一些实施例采用的多酶体系拆分法生产L-草铵膦的反应式。
图2-1示例性显示了用重组大肠杆菌E.coli BL21(DE3)/pET-28a-APH1单酶以及E.coli BL21(DE3)/pET-28a-APH1&E.coli BL21(DE3)/pET-28a-DAAO催化效率对比柱状图，图2-2示例性显示了E.coli BL21(DE3)/pET-28a-APH1和E.coli BL21(DE3)/pET-28a-DAAO拆分消旋PPT的反应进程曲线。
图3示例性显示了用重组大肠杆菌E.coli BL21(DE3)/pET-28a-EN3和E.coli BL21(DE3)/pET-28a-PDC还原胺化PPO合成L-PPT的反应进程曲线。
图4示例性显示了APH1-DAAO构建体。
图5示例性显示了EN3-PDC构建体。
图6示例性显示了用重组大肠杆菌共表达菌株E.coli BL21(DE3)/pCDFduet-1-APH1-DAAO拆分消旋PPT的反应进程曲线。
图7示例性显示了用重组大肠杆菌共表达菌株E.coli BL21(DE3)/pCDFduet-1-EN3-PDC还原胺化PPO合成L-PPT的反应进程曲线。
图8示例性显示了双菌多酶一锅两步法去消旋化制备L-草铵膦的反应进程曲线。
具体实施方式
实施例
材料和方法
基因工程所用材料和试剂：基因组提取试剂盒、质粒提取试剂盒、DNA纯化回收试剂盒购自康宁生命科学(吴江)有限公司；一步克隆试剂盒是诺唯赞有限公司(Vazyme)的
Figure PCTCN2021139009-appb-000001
MultiS One Step Cloning Kit；E.coli DH5α、E.coli BL21(DE3)、质粒pET-28a(+)、pCDFduet-1载体等购自上海旭冠生物科技 发展有限公司；DNA marker、低分子量标准蛋白、蛋白胶等购自北京GenStar有限公司；过氧化氢酶购自宁夏夏盛实业集团有限公司，商品编号为CAT-400。以上试剂使用方法参考商品说明书。
序列合成，序列测序工作由杭州擎科梓熙生物技术有限公司完成。
催化工艺所用试剂2-羰基-4-[羟基(甲基)膦酰基]丁酸(简称PPO)，D,L-草铵膦购自永农生物科学有限公司；其他常用试剂购自国药集团化学试剂有限公司。
通过高效液相色谱(HPLC)检测反应的进行，并对PPO进行分析。HPLC分析方法为：色谱柱/PBr；柱温/30℃；流速/0.8mL/min；检测波长/210nm；流动相：50mM(NH4)2HPO4，加入12％的乙腈。
通过手性HPLC分析方法检查草铵膦的两个构型含量，手性HPLC分析方法为：色谱柱/OA-5000L；流动相/0.5％五水硫酸铜；检测波长/254nm；流速/0.7mL/min；柱温/35℃。
实施例1：工程菌菌体的培养
将工程菌重组大肠杆菌E.coli BL21(DE3)/pET-28a-APH1、E.coli BL21(DE3)/pET-28a-DAAO、E.coli BL21(DE3)/pET-28a-EN3、E.coli BL21(DE3)/pET-28a-PDC经平皿划线活化后，挑单菌落接种至含有50μg/mL卡那霉素的10mL LB液体培养基中，37℃震荡培养10h。按2％的接种量转接至50mL同样含有50μg/mL卡那霉素的LB液体培养基中，37℃震荡培养至OD600达到0.8左右时，加入终浓度为0.5mM的IPTG，28℃下震荡培养12h。培养结束后，将培养液8000rpm离心10min，弃上清，收集菌体，放到-80℃超低温冰箱中保存，待用。
实施例2：酶序列合成和菌株构建
将来源于Pseudarthrobacter chlorophenolicus的注释为(R)-转氨酶(APH1)的序列(氨基酸序列为SEQ ID NO.1所示，核苷酸序列为SEQ ID NO.2所示)进行全基因合成后，插入表达质粒pET-28a(+)得到pET28a-APH1。测序验证无误后将pET28a-APH1转入表达宿主大肠杆菌E.coli BL21(DE3)中用于后续重组酶的表达。
将来源于Rhodotorula sp.CCFEE 5036的注释为D-氨基酸氧化酶(DAAO)的序列(氨基酸序列为SEQ ID NO.3所示，核苷酸序列为SEQ ID NO.4所示)进行全基因合成后，插入表达质粒pET-28a(+)得到pET28a-DAAO。测序验证无误后将pET28a-DAAO转入表达宿主大肠杆菌E.coli BL21(DE3)中用于后续重组酶的表达。
将来源于Paraburkholderia phymatum STM815注释为(S)-转氨酶(EN3)的序列(氨基酸序列为SEQ ID NO.7所示，核苷酸序列为SEQ ID NO.8所示)进行全基因合成后，插入表达质粒pET-28a(+)得到pET28a-EN3。测序验证无误后将pET28a-EN3转入表达宿主大肠杆菌E.coli BL21(DE3)中用于后续重组酶的表达。
将来源于Zymobacter palmae注释为丙酮酸脱羧酶(PDC)的序列(氨基酸序列为SEQ ID NO.5所示，核苷酸序列为SEQ ID NO.6所示)进行全基因合成后，插入表达质粒pET-28a(+)得到pET28a-PDC。测序验证无误后将pET28a-PDC转入表达宿主大肠杆菌E.coli BL21(DE3)中用于后续重组酶的表达。
实施例3：用重组大肠杆菌E.coli BL21(DE3)/pET-28a-APH1和E.coli BL21(DE3)/pET-28a-DAAO拆分消旋PPT
(i)重组大肠杆菌E.coli BL21(DE3)/pET-28a-APH1单酶催化体系实验：30ml的反应体系中含有500mM D,L-PPT，250mM丙酮酸，和100mM磷酸盐缓冲液，用氨水将反应体系的pH调节至8.0，加入重组大肠杆菌E.coli BL21(DE3)/pET-28a-APH1 30g/L干细胞。反应条件为：温度30℃，转速250rpm。反应10h取样(100μl)，加入900μl去离子水，加热终止反应。通过HPLC检测D-PPT的转化情况，反应结果如图2-1所示。
(ii)重组大肠杆菌E.coli BL21(DE3)/pET-28a-APH1和E.coli BL21(DE3)/pET-28a-DAAO双酶催化体系实验：30ml的反应体系中含有500mM D,L-PPT，250mM丙酮酸，和100mM磷酸盐缓冲液，用氨水将反应体系的pH调节至8.0，加入重组大肠杆菌E.coli BL21(DE3)/pET-28a-APH1和E.coli BL21(DE3)/pET-28a-DAAO各30g/L干细胞。反应条件为：温度30℃，转速250rpm。反应10h取样(100μl)，加入900μl去离子水，加热终止反应。通过HPLC检测D-PPT的转化情况，反应结果如图2-1所示。
结果显示，在反应体系中添加DAAO能够明显提高APH1转化D-PPT的效率。
(iii)D-PPT拆分进程曲线实验：30ml的反应体系中含有500mM D,L-PPT，10mM丙酮酸，4000U/mL过氧化氢酶和100mM磷酸盐缓冲液，用氨水将反应体系的pH调节至8.0，加入重组大肠杆菌E.coli BL21(DE3)/pET-28a-APH1和E.coli BL21(DE3)/pET-28a-DAAO各30g/L干细胞。反应条件为：温度30℃，转速250rpm。每隔一段时间取样(100μl)，加入900μl去离子水，加热终止反应。通过HPLC检测D-PPT的转化情况，反应进程曲线如图2-2所示。
结果显示，随着时间的推移，D-PPT转化率逐渐升高，60h内反应完成，底物转化率大于99.9％。
实施例4：用重组大肠杆菌E.coli BL21(DE3)/pET-28a-EN3和E.coli BL21(DE3)/pET-28a-PDC还原胺化PPO合成L-PPT
30ml的反应体系中含有500mM PPO，1.2M L-丙氨酸，100mM磷酸盐缓冲液，用氨水将反应体系的pH调节至8.0，加入重组大肠杆菌E.coli BL21(DE3)/pET-28a-EN3和pET-28a-PDC各30g/L干细胞。反应条件为：温度30℃，转速250rpm。每隔一段时间取样(100μl)，加入900μl去离子水，加热终止反应。通过HPLC检测PPO的转化情况，反应进程曲线如图3所示。
结果显示，随着时间的推移，PPO转化率逐渐升高，44h内反应完成，底物转化率大于85％。
实施例5：共表达菌株的构建
一、含(R)-转氨酶和D-氨基酸氧化酶的共表达菌株的构建
将实施例2中使用的APH1基因序列通过一步克隆试剂盒连接到多克隆位点载体pCDFduet-1上，酶切位点为HindIII和XhoI，一步克隆引物为C1-F和C1-R(表1)，构建得到质粒pCDFduet-1-APH1。再在pCDFduet-1-APH1质粒的基础上，通过所述一步克隆试剂盒将实施例2中使用的DAAO片段连接到多克隆位点载体pCDFduet-1第二个克隆位点上，酶切位点为NdeI和XhoI，一步克隆引物为C2-F和C2-R(表1)，构建得到质粒pCDFduet-1-APH1-DAAO，将 质粒转化入菌株E.coli BL21(DE3)得到共表达菌株E.coli BL21(DE3)/pCDFduet-1-APH1-DAAO。图4显示了APH1-DAAO构建体。
二、含(S)-转氨酶和丙酮酸脱羧酶的共表达菌株构建
将实施例2中使用的EN3基因通过一步克隆试剂盒连接到多克隆位点载体pCDFduet-1上，酶切位点为NcoI和HindⅢ，一步克隆引物为C3-F和C3-R(表1)，构建得到质粒pCDFduet-1-EN3。再在pCDFduet-1-EN3质粒的基础上，通过所述一步克隆试剂盒将PDC连接到多克隆位点载体pCDFduet-1第二个克隆位点上，酶切位点为NdeI和EcoRI，一步克隆引物为C4-F和C4-R(表1)，构建得到质粒pCDFduet-1-EN3-PDC，将质粒转入菌株E.coli BL21(DE3)得到共表达菌株E.coli BL21(DE3)/pCDFduet-1-EN3-PDC。图5显示了EN3-PDC构建体。
表1：克隆引物序列
引物 序列
C1-F CCCAAGCTTAAGGAGATATACATATGACCTCTCCCGCTTCCGT
C1-R CCGCTCGAGCTATTGGATTCCGGCGTAAAGC
C2-F CCCAAGCTT5ATGCACAGCCAGAAACGCGTAGTTGTTCTGGGTAG
C2-R CCGCTCGAG TTACAGTTTGCTTTCGCGTGCTGCGCCATGATAAC
C3-F CCGGAATTCAAGGAGATATACATATGAAGAATGCTGAACTGAAGAGCC
C3-R ATAAGAATGCGGCCGCTCAGGCCGCTACGCCAAC
C4-F CCGGAATTCATGTATACCGTTGGTATGTACTTGGCAGAACGCCT
C4-R ATAAGAATGCGGCCGCTTACGCTTGTGGTTTGCGAGAGTTGGTAGCTGCTA
实施例6：用重组大肠杆菌共表达菌株E.coli BL21(DE3)/pCDFduet-1-APH1-DAAO拆分消旋PPT
按照实施例5(一)的方法构建并培养能够表达(R)-转氨酶和D-氨基酸氧化酶的共表达菌株E.coli BL21(DE3)/pCDFduet-1-APH1-DAAO。
30ml的反应体系含有500mM D,L-PPT，10mM丙酮酸，4000U/mL过氧化氢酶，100mM磷酸盐缓冲液，用氨水调节将反应体系的pH调节至8.0，加入共表达菌株E.coli BL21(DE3)/pCDFduet-1-APH1-DAAO 30g/L干细胞。反应条件为：温度30℃，转速250rpm。每隔一段时间取样(100μl)，加入900μl去离 子水，加热终止反应。通过HPLC检测D-PPT的转化情况，反应进程曲线如图6所示。
结果显示，随着时间的推移，D-PPT转化率逐渐升高，16h内反应完成，底物转化率大于99.9％。
实施例7：用共表达菌株E.coli BL21(DE3)/pCDFduet-1-EN3-PDC还原胺化PPO合成L-PPT
按照实施例5(二)的方法构建和培养能够表达(S)-转氨酶和丙酮酸脱羧酶的共表达菌株E.coli BL21(DE3)/pETduet-1-EN3-PDC，离心收集菌体细胞。
30ml反应体系中含有500mM PPO，1.2M L-丙氨酸，100mM磷酸盐缓冲液，用氨水将反应体系的pH调节至8.0，加入共表达菌株E.coli BL21(DE3)/pCDFduet-1-EN3-PDC 30g/L干细胞。反应条件为：温度30℃，转速250rpm。每隔一段时间取样(100μl)，加入900μl去离子水，加热终止反应。通过HPLC检测PPO的转化情况，反应进程曲线如图7所示。
结果显示，随着时间的推移，PPO转化率逐渐升高，EN3-PDC在30h内完成反应，底物转化率大于99.9％。
实施例8：双菌多酶一锅两步法去消旋化制备L-草铵膦
按照实施例5(一)的方法构建和培养能够表达(R)-转氨酶和D-氨基酸氧化酶的共表达菌株E.coli BL21(DE3)/pCDFduet-1-APH1-DAAO。按照实施例5(二)的方法构建和培养能够表达(S)-转氨酶和丙酮酸脱羧酶的共表达菌株E.coli BL21(DE3)/pETduet-1-EN3-PDC，离心收集菌体细胞。
在1L反应器中，加入600mL pH＝8的磷酸盐缓冲液，温度设定为30℃，加入500mM D,L-PPT、消泡剂泡敌、10mM丙酮酸，过氧化氢酶1％v/v(4000U/ml)，30g/L E.coli BL21(DE3)/pCDFduet-1-APH1-DAAO菌体，通入空气，通气量为2L/min，反应14小时；75℃加热30min，再加入30g/L E.coli BL21(DE3)/pETduet-1-EN3-PDC菌体和300mM L-丙氨酸，用氨水控制pH为8，反应15小时。液相检测D-PPT为0mM，D-PPT转化率为99.9％，PPO为2mM，L-PPT为398mM，产品草铵膦的e.e.值为99.9％。反应进程曲线如图8所示。

Claims (21)

  1. 一种制备L-草铵膦的方法，其包括：
    a)在(R)-转氨酶和氨基受体的存在下，使D,L-草铵膦发生转氨反应得到2-羰基-4-[羟基(甲基)膦酰基]丁酸和氨基受体的加氨基产物；和
    b)在(S)-转氨酶和氨基供体的存在下，使步骤a)中得到的2-羰基-4-[羟基(甲基)膦酰基]丁酸发生转氨反应得到L-草铵膦和氨基供体的脱氨基产物。
  2. 根据权利要求1所述的方法，其中步骤a)中另外存在能够使所述氨基受体的加氨基产物转化为所述氨基受体的催化酶。
  3. 根据权利要求1或2所述的方法，其中步骤b)中另外存在能够除去所述氨基供体的脱氨基产物的催化酶。
  4. 根据前述权利要求中任一项所述的方法，其中所述步骤a)之后和步骤b)之前还包加热步骤，所述加热步骤使步骤a)的反应混合物中的酶灭活。
  5. 根据前述权利要求中任一项所述的方法，其中所述方法在同一反应器中进行。
  6. 根据前述权利要求中任一项所述的方法，其中步骤a)中的所述氨基受体为丙酮酸，所述氨基受体的加氨基产物为D-丙氨酸。
  7. 根据前述权利要求中任一项所述的方法，其中步骤b)中的所述氨基供体为L-丙氨酸，所述氨基供体的脱氨基产物为丙酮酸。
  8. 根据权利要求2至7中任一项所述的方法，其中在步骤a)中能够使所述氨基受体的加氨基产物转化为所述氨基受体的所述催化酶为D-氨基酸氧化酶。
  9. 根据权利要求3至8中任一项所述的方法，其中在步骤b)中能够除去所述氨基供体的脱氨基产物的所述催化酶为丙酮酸脱酸酶。
  10. 根据前述权利要求中任一项所述的方法，其中步骤a)的反应中另外存在过氧化氢酶。
  11. 根据前述权利要求中任一项所述的方法，其中所述(R)-转氨酶、(S)-转氨酶、能够使所述氨基受体的加氨基产物转化为所述氨基受体的催化酶、能够除去所述氨基供体的脱氨基产物的催化酶和所述过氧化氢酶的形式各自独立地选 自：部分纯化的酶；无细胞提取物或粗细胞提取物；液体、粉末或固定形式；含有酶的可透化处理的细胞、完整细胞或完整发酵液、冻干细胞或其任何组合。
  12. 根据权利要求2-10中任一项所述的方法，其中所述方法的步骤a)包括：
    a)在共表达(R)-转氨酶和能够使所述氨基受体的加氨基产物转化为所述氨基受体的所述催化酶的第一重组微生物和氨基受体的存在下，使D,L-草铵膦发生转氨反应得到2-羰基-4-[羟基(甲基)膦酰基]丁酸并使得到的所述加氨基产物转化为所述氨基受体。
  13. 根据前述权利要求中任一项所述的方法，其中所述方法的步骤b)包括：
    b)在共表达(S)-转氨酶和能够除去所述氨基供体的脱氨基产物的催化酶的第二重组微生物和氨基供体的存在下，使2-羰基-4-[羟基(甲基)膦酰基]丁酸发生转氨反应得到L-草铵膦并除去得到的所述脱氨基产物。
  14. 根据前述权利要求中任一项所述的方法，其中所述第一重组微生物和第二重组微生物各自独立地选自：酿酒酵母(Saccharomyces cerevisiae)、解脂耶氏酵母(Yarrowia lipolitica)、克鲁斯假丝酵母(Candida krusei)、东方伊萨酵母(Issatchenkia orientalis)或大肠杆菌(Escherichia coli)。
  15. 根据权利要求12-14中任一项所述的方法，其中，以湿菌体重量计，所述第一重组微生物的添加量为5-200g湿菌体/L反应液，或者，以干菌体重量计，所述第一重组微生物的添加量为1-50g干菌体/L反应液。
  16. 根据权利要求13-15中任一项所述的方法，其中，以湿菌体重量计，所述第二重组微生物的添加量为5-200g湿菌体/L反应液，或者，以干菌体重量计，所述第二重组微生物的添加量为1-50g干菌体/L反应液。
  17. 根据前述权利要求中任一项所述的方法，其中步骤a)和b)的反应在反应缓冲液中进行，优选地，所述反应缓冲液是pH为8-9的磷酸氢二钠-磷酸二氢钠缓冲液。
  18. 根据前述权利要求中任一项所述的方法，其中在步骤a)中，反应开始时氨基受体与D,L-草铵膦的摩尔比为1:500-1:5。
  19. 根据前述权利要求中任一项所述的方法，其中在步骤b)中，反应开始时氨基供体与底物的摩尔比为1:2-5:1。
  20. 根据前述权利要求中任一项所述的方法，所述步骤a)和步骤b)的转氨反应的温度为30-45℃，时间各为14-48h。
  21. 根据前述权利要求中任一项所述的方法，其中步骤a)和步骤b)的反应在辅酶磷酸吡哆醛的存在下进行，优选地磷酸吡哆醛与底物的摩尔比为1:10-1:200。
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